Modifying Remdesivir Prodrug Design to Enhance the Active Metabolite Accumulation in the Lung (Resubmission)

摘要 尽管瑞德西韦是FDA批准用于治疗COVID-19患者的少数药物之一， 大部分患者对治疗没有充分反应。Remdesivir形成为2- 核苷类似物GS-441524的乙基丁酯前药，以克服 核苷，并绕过GS-441524活化所需的限速单磷酸化代谢。 Remdesivir在人血浆中广泛水解，引起了人们的担忧， Remdesivir可以以其完整的前药形式到达肺部。因此，与前药设计相关的益处 可能无法用于治疗COVID-19。另一个局限性是，瑞德西韦仅可用于 静脉注射剂型由于其强烈的首过效应。由于这些限制，一些 研究人员主张使用母体化合物GS-441524作为Remdesivir的替代品来治疗 新冠肺炎患者。然而，目前尚不清楚GS-441524的细胞渗透性差如何影响细胞内 药物蓄积和所需的限速单磷酸化步骤可能影响的程度 GS-441524激活。在瑞德西韦的开发过程中，合成了几种GS-441524酯前药 并评价其血浆稳定性和体外抗病毒活性。Remdesivir被选为药物 由于其相对于其他前药在抗埃博拉病毒方面的上级活性， 几种非肺细胞系。其中，GS-441524的异丙酯前体显示出较低的抗病毒活性 但血浆稳定性比Remdesivir高得多。鉴于所有前药具有相同的活性 由于前体药物的抗病毒活性与代谢产物的差异有关，因此受试前体药物之间的抗病毒活性差异可能归因于它们的差异 在细胞内积累和激活。现有的证据表明，羧酸酯酶1（CES 1）和 组织蛋白酶A（CatA）参与酯核苷前药的活化，并且CES 1和CatA都是 在人肺中高度表达。因此，异丙基-GS-441524可在肺中有效活化， 从而发挥其抗SARS-CoV-2的作用。此外，异丙基-GS-441524在水中具有出色的稳定性 血浆、肝和肠可改善肺中细胞内药物蓄积， 化合物被开发成口服剂型。在本项目中，我们将进行药代动力学（PK） 在小鼠中进行的比较异丙基-GS-441524、Remdesivir和GS-441524之间的活化和处置的研究。 441524在各种组织中。我们还将确定这三种化合物在人体内的激活率。 肺和其他PK相关器官，并鉴定和表征负责前药的水解酶 activation.此外，我们还将使用体外抗SARS-CoV-2活性评价三种化合物的抗SARS-CoV-2活性。 模型该项目将提供证据支持异丙基-GS-441524优于Remdesivir 和GS-441524用于治疗COVID-19。此外，研究组织特异性酯前药激活水解酶， 这将为未来开发更有效的核苷前药提供线索。

Abstract Although remdesivir is among few medications approved by the FDA for treating patients with COVID-19, a significant portion of patients did not respond adequately to the treatment. Remdesivir is formed as a 2- ethylbutyl ester prodrug of the nucleoside analog GS-441524 to overcome the poor cell permeability of nucleosides and bypass the rate-limiting mono-phosphorylation metabolism required for GS-441524 activation. Remdesivir is extensively hydrolyzed in human plasma, raising the concerns that only a small portion of remdesivir can reach the lung in its intact prodrug form. Thus, the benefits associated with the prodrug design may be unattainable for the treatment of COVID-19. Another limitation is that remdesivir is only available in an intravenous injection dosage form due to its intensive first-pass effect. Because of those limitations, some investigators advocate for using the parent compound GS-441524 as an alternative to remdesivir to treat COVID-19 patients. However, it remains unknown how GS-441524's poor cell permeability affects intracellular drug accumulation and the extent to which the required rate-limiting mono-phosphorylation step could affect GS-441524 activation. During remdesivir development, several GS-441524 ester prodrugs were synthesized and evaluated for their plasma stability and in vitro antiviral activity. Remdesivir was chosen as the drug candidate for further development because of its superior anti-Ebola activity relative to other prodrugs in several non-lung cell lines. Among those, the isopropyl ester prodrug of GS-441524 showed lower antiviral potency but much greater plasma stability than remdesivir. Given that all the prodrugs have the same active metabolite, the antiviral activity differences among the tested prodrugs are likely attributed to their differences in intracellular accumulation and activation. Existing evidence suggests that carboxylesterase 1 (CES1) and cathepsin A (CatA) are involved in the activation of ester nucleoside prodrugs, and both CES1 and CatA are highly expressed in the human lung. Thus, isopropyl-GS-441524 could be efficiently activated in the lung and consequently exert its anti-SARS-CoV-2 effect. Moreover, the excellent stability of isopropyl-GS-441524 in plasma, liver, and intestine could improve the intracellular drug accumulation in the lung and allow the compound to be developed into an oral dosage form. In this project, we will conduct a pharmacokinetics (PK) study in mice to compare the activation and disposition between isopropyl-GS-441524, remdesivir, and GS- 441524 in various tissues. We will also determine the activation rates of the three compounds in the human lung and other PK-related organs and identify and characterize the hydrolases responsible for the prodrug activation. Furthermore, we will evaluate the anti-SARS-CoV-2 activity of the three compounds using an in vitro model. The project will provide evidence supporting that isopropyl-GS-441524 is advantageous over remdesivir and GS-441524 for treating COVID-19. Moreover, studying tissue-specific ester prodrug-activating hydrolases will shed light on the future development of more effective nucleoside prodrugs.